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BULLETIN OF THE 



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No. 70 



Contribution from the Bureau of Animal Industry, A. D. Melvin, Chief 
April 15, 1914. 




(PROFESSIONAL PAPER.) 



IMMUNIZATION TESTS WITH GLANDERS VACCINE. 

By John R. Mohleh and Adolph Eichhorn, 
Pathological Division, Bureau of Animal Industry 

INTRODUCTORY. 

Among the diseases of horses with which the vetermary authori- 
ties arc concerned glanders is probably the most important, and 
unless strict measures for its control are enforced the tendency of 
the disease is to spread more or less rapidly. This fact is due to the 
character of the disease, to the prevailing methods of carmg for 
horses, and, probably more hnportant than all, to the frequent 
latent existence of the disease in apparently healthy animals. The 
destruction of all infected animals has been accepted as a matter of 
course in all civilized countries, and owing to the dangerous character 
of the disease and the possibility of transmission to man, this action 
appears to be the sanest and most reasonable procedure m its control. 
On the other hand, the possibility of a method of immunization of 
healthy animals is worthy of consideration and would be of great 
advantage. 

Ever since the discovery of maUein as a diagnostic agent for glan- 
ders, experunents have been conducted by various mvestigators 
relative to its immunizing and curative value. Many favorable 
reports have been made by veterinarians of the results obtained. 
On the contrary, others appear to have had no satisfaction from its use. 
[Since it has been proved that cases of glanders may recover it is 
rather difficult to establish the value of the immunizing agents as to 
[theu* action on the disease. Fortunately, we now possess a means 
! by which the presence of immune bodies can be demonstrated m the 
animal upon which attempts at immunization are made. With the 
serological tests at our command we may control to some extent the 
action of an immunizing substance and observe how long the immune 
bodies are present in an animal receiving immunization treatment. 

24133°— 14 .. 

Morwgra^ 



2 BULLETIN 70, U. S. DEPARTMENT OF AGRICULTURE. Cq> \ V ^ 

It is unfortunate, however, that the demonstration of immune 
bodies does not indicate the degree of immunity in the animals. 

We may obtain in glanders immunization an agglutination value 
of 1 to 5,000 or over or a complement fixation with 0.02 of a cubic 
centimeter of serum which may contmue for a period of several 
months, yet this same animal, which apparently is supphed with a 
great amount of immune bodies, can be readily infected with glanders 
bacilli. Therefore, in tests undertaken for establislung the degree 
of immunity against glanders in the horse, it is necessary to expose 
the injected animals to an hifection such as occurs under natural 
conditions. Observations of such animals as to the clinical appear- 
ance of the disease and periodical ophthahnic tests with maUein are 
the methods by which the most accurate results of the immunization 
tests can be obtamed. Serum tests in these cases are of little value, 
as they invariably demonstrate immune bodies or antibodies in the 
immunized animals, and since even small quantities of mallein 
injected mto a horse are sufficient to produce antibodies which remain 
for 3 or 4 weeks. 

PREVIOUS RESULTS WITH VARIOUS IMMUNIZING AGENTS. 

Curative results from maUem were reported by Leclainche, Hueppe, 
Nocard, Johne, and Wladimiroff, while its immunizing value against 
glanders was studied by Schindelka, McFadyean, and Semmer, but 
the results were unsatisfactory. Taking into consideration the litera- 
ture at our command and drawing conclusions from the results ob- 
tained, it appears that mallein possesses very little immunizing value 
and no great benefit can be expected from its use as a curative 
agent. 

Other investigators attempted to immunize horses and other 
animals against glanders with the use of killed glanders bacilli and 
the literature contains some favorable results from this method of 
immunization. The preparations which were employed for this pur- 
pose were in most instances suspensions of glanders bacilli killed by 
heat. Of the various products which have been prepared and are at 
the present time used to a limited extent for the immunization of 
glanders, ''farase," so termed by Levy, Blumenthal, and Marxer, 
gives apparently the best results. It is prepared by kiUing glanders 
bacilli with 80 per cent glycerin or 10 per cent urea. The baciUi are 
then dried and the substance is used in that condition for the immu- 
nization. It does not contain living bacteria. Favorable results were 
obtained with farase by Bautz and Machodin, and by Dediuhn. The 
results of Dediuhn are probably the most remarkable, since he reports 
that on an estate where previous to immunization 276 glandered ani- 
mals had been destroyed, he injected 303 animals and after one year and 

V), OF Q. 
APR 2? .914 



IMMUNIZATION TESTS WITH GLANDEES VACCINE. 3 

four months not a single case of glanders developed, although in the 
meanwhile 14 cases of glanders developed among 300 nonimmunized 
animals. 

Bautz and Machodin subjected farase to various tests to establish 
its immunizing value. Their results on guinea pigs, cats, and horses 
were very satisfactory. Guinea pigs which were given two injections 
of farase resisted six weeks later an intraperitoneal infection with 
1/2500 and 1/5000 mg. of glanders bacilli. Of six horses which 
received two immunizing injections of farase, two were given 1/2500 
mg. of glanders bacilli subcutaneously, two received 1/500 mg. of 
glanders baciUi per os, and two were exposed with the other animals 
45 days after the second injection. For each of the groups one check 
was used. Post-mortem examination of the check animals four to 
five weeks after the infection showed typical glanders, while the two 
immunized animals which received subcutaneous injections of 
glanders bacilli failed to show any lesions of the disease. No record 
was obtained of the four remaining immunized animals, as they were 
turned over to another laboratory for study of the duration of immu- 
nity in these horses. 

One of the recent works on the immunization of glanders was pub- 
lished by Zurkan, who studied the formation of specific antibodies in 
the blood of horses under the action of glanders antigens. He con- 
cludes that of various antigens such as farase, killed glanders bacilli, 
mallein, and malleo-aggressin, the fu'st and the last (farase and malleo- 
aggressin) proved most active in the production of immune bodies. 
The degree of immunity in the animals was estabhshed by Zurkan 
from the comparative results of the serological reactions he obtained 
with the complement-fixation, agglutination, precipitation, and 
opsonic tests. Since there were no practical tests made on these 
animals, his statement that malleo-aggressin may be used for the 
immunization of horses against glanders can not be accepted as 
conclusive. 

At the meeting of the American Veterinary Medical Association in 
Indianapolis, MacKellar presented his conclusions on the protective 
effect of glanders vaccine. The proportion of infections in the stables 
where these outbreaks occurred, as indicated by the agglutination 
test, is astonishing. As there is no mention made in the article of the 
time the agglutmation tests were applied subsequent to the mallein 
test, it suggests that the large proportion of reactors to the agglutina- 
tion test were the result of the mallein injection and not due to the 
presence of the infection. If this be true, then the effect of the 
vaccine remains indefinite and the control of the disease must be 
accredited to the other precautions which were observed. At best it 
will require several years before the value of any method of immu- 
nization can be satisfactorily estabhsl^ed. 



4 BULl.ETIN '70, V. S. DEPARTMENT OF AGRICULTURE. 

EXPERIMENTS WITH DRIED GLANDERS BACILLI. 

The New York City board of health has been conducting immu- 
nizing experiments with a vaccine prepared in their laboratory, con- 
sisting of a suspension of dried glanders bacilli. Each cubic centi- 
meter of the suspension contains 2 mg. of dried bacUli. Through the 
courtesy of Dr. William H. Park, director of the laboratory, a suffi- 
cient quantity of such vaccine was obtained for conducting a series of 
investigations relative to the possibility of conferring immunity to 
animals injected wdth this vaccine. 

GUINEA-PIG EXPERIMENTS. 

The experiments were made on guinea pigs and on horses. Twenty 
guinea pigs, about 600 grams in weight, were divided into 4 groups, 
4 pigs of each group receiving three immunizing injections of a definite 
amount of vaccine at intervals of one week. The size of the doses 
and other details are presented in Table 1. After the conclusion of 
these vaccinations one pig from each group was subjected to infection 
with suspensions of glanders bacilli. These injections with infectious 
material were administered at various intervals. In aU instances the 
same strain of glanders baciUi was used for the infections. The fifth 
pig in each group was not vaccinated, but served as a check, receiving 
only a corresponding quantity of glanders bacilh. 

The results of these guinea-pig tests showed that there was not a 
sufiicient increased resistance among the vaccinated guinea pigs to 
warrant any hopes of successful immunization by this method. It is 
to be regretted, however, that in the infection of these pigs probably 
too large a quantity of glanders baciUi was used. On the other hand, 
it would appear that if there had been any appreciable immunity 
present in the vaccinated guinea pigs they would have manifested it 
by a greater resistance against the infection. 



IMMUNIZATION TESTS WITH GLANDEKS VACCINE. 



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6 BULLETIN 70, U. S. DEPARTMENT OF AGEICULTURE. 

EXPERIMENTS ON HORSES. 

In the experiments conducted on horses, 17 animals were used, 
which were purchased on the open market. Most of the animals 
were aged, but otherwise in fair condition. AH the horses w^ere 
subjected to the agglutination, complement-fixation, and the ophthal- 
mic mallein tests, prior to the vaccination. All of them proved free 
from glanders on all the tests. Since the amount of vaccine to be 
injected for immunizing purposes has not been established, it was 
deemed advisable to employ varying quantities for the injections, 
and in order to determine the resistance of the animals against 
infection durmg and after the vaccination they were subjected to 
exposure at different times during the investigation. 

The smallest amount of the suspension used for the vaccination 
was the quantity recommended by the New York City Board of 
Health, viz, 1, 3, and 5 c. c. per injection, while the largest amount 
any of the horses received was 4, 8, and 12 c. c, respectively. Two 
of the vaccinated horses received an infection on the nasal mucosa 
with glanders bacilli, taken up on the end of a platinum loop, one 
week after the last vaccination. Both of these horses promptly 
developed glanders and one of them. No. 102, died of an acute form 
of the disease 21 days after the infection. Thus, there appeared to 
be no resistance, or at least no increased resistance, against artificial 
infection. 

To establish the resistance of the vaccinated animals against 
contact infection a corral was built where all the animals, including 
two artificially infected glanders cases, were kept. They were fed in 
common feed boxes and were watered from a common trough. Only 
one hayrack was used for all animals. Simultaneously with this 
exposure a stable with three stalls was likewise used for exposing 
the horses. The construction of the stalls in this stable was such 
that the animal in the center could reach to the feed boxes of either 
of the horses in the side stalls. The horse placed in the center was 
a good discharging case of cHnical glanders, whereas the horses 
placed in the side stalls were either two immimized animals or two 
controls, all of wliich were given one week's exposure with this 
infected horse. This was accomplished by changing the horses in 
the two side stalls every week, and bringing in two others from the 
corral, so as to make the exposure as uniform as possible in all ani- 
mals, including the checks. The conditions of exposure were appar- 
ently severe, yet they did not exceed the exposure wliich occurs in 
the stables of large cities where the sanitary conditions are very 
poor and where poor light and ventilation afford a splendid oppor- 
tunity for the propagation of the disease. In fact, the exposure in 
the corral was rather slight, since the sunlight no doubt had a 
destructive influence on the infection. 



IMMUNIZATION TESTS WITH GLANDEES VACCINE. 7 

All animals were subjected periodically to clinical examinations 
and only four of the vaccinated horses developed signs of the disease 
up to the conclusion of this experiment, although some of them were 
exposed since May 16. Horse No. 99, which received 4 immunizing 
injections and was exposed to a discharging case of glanders in the 
stable, died 15 days after the exposure from acute broncho-pneu- 
monia rhalleosa. 

In order to determine whether any of the vaccinated horses were 
infected with the latent form of the disease, all were subjected July 
23 to the ophthalmic test. This gave sm-prising results. Two of 
the vaccinated animals gave a marked reaction (P + -h + ) . A 
similar reaction was also obtamed in the affected horses used for 
exposui'e, while of the two check animals wliich were not vaccinated 
but had been exposed to a similar extent as the vaccinated animals, 
only one responded to the test; the other check animal failed to give 
any reaction. One month later all horses in the experiment were 
again subjected to the ophthalmic test. The results were the same 
as on the previous test, but it was noted that the intensity of the 
reaction was not as pronounced as in the first test. The inflammation 
and amount of purulent discharge were somewhat less than in the 
previous test. This observation coincides with that of Meyer, who 
states that after several eye tests in positive cases of glanders the 
degree of the reaction becomes less distinct. 

The detailed account of the results of the immunizing tests in 
horses is given in Table 2. 



BULLETIN 70, U. S. DEPARTMENT OF AGEICULTUEE. 






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IMMUNIZATION TESTS WITH GLANDERS VACCINE. 9 

AGGLUTINATION AND COMPLEMENT-FIXATION TESTS. 

Ill order to study the effect of tlie immuiiizing injections on the 
serum tests, the blood of the horses in this experiment was subjected 
to the agglutination and complement-fixation tests from the time of 
the first injection until the conclusion of the work. It was found 
that the agglutination value of the serum of the vaccinated horses 
as a rule increased from the tliird day after the first vaccination and 
continued to rise for a time. A decrease was again noted from two 
to fom' Aveeks after the last vaccination and appeared practically 
normal after six weeks to two months. A complement fixation with 
the sera of the A^accinated horses Avas obtained from the seventh to 
the ninth day after the fii'st vaccination and they continued to giA^e 
positiA'e fixations from two to three months after the last vaccination. 

These negatiA-e serological results Avhich folloAved the positiA^e reac- 
tions due to the injected vaccine, appeared only in the animals Avhich 
gaA^e no reaction to the ophthalmic test, Avhile the blood of those A'acci- 
nated horses wliich gave a positive reaction to the eye test continued 
to give a positive fixation until they had been destroyed and proA-^ed 
to be affected Avitli the disease. The same condition was observed 
in the animals wliich had been artificially infected with glanders. 

The serological results from these investigations appear to have a 
great significance AA-ith reference to the immunity produced by the 
injection of dead glanders bacilU. The fact that the demonstration 
of the presence of immune bodies in the vaccinated horses ceased 
entirely in two or three months from the last A^accination would 
indicate that after the lapse of such a time the animals have A^ery 
little or no immunity against the disease. This is further substan- 
tiated also by the agglutmation value of the sera returning to the 
normal level. As a matter of fact, preAaous investigations carried 
out by Dr. Buck, of this laboratory, showed that one or two sub- 
cutaneous injections of mallein Avill give a complement fixation which 
may last from one to tAvo months. The agglutination A^alue of the 
serum of such animals is also markedly influenced by subcutaneous 
malleinization. The serum reaction of horses folloAving the sub- 
cutaneous injections of mallein is giA^en in detail in Table 4. From 
this it seems that a mallein injection has almost the same action on 
the production of immune bodies in a horse as killed glanders bacilH. 
Table 3 indicates the results obtained with the agglutination and com- 
plement-fixation tests in the animals used in tliis uivestigation. 

On August 20 two A^accinated horses and one check animal wliich 
gave positiA^e results to the eye test Avere destroyed, and in ail three 
animals marked pulmonary glanders was observed. Horse No. 105 
showed the presence of glanders nodules in the lungs in very great 
numbers, some of which were of the size of a walnut. In the two 
other cases, A\-hile the nodules Avere A^ery numerous and from their 



10 BULLETIN 70, U. S. DEPAETMENT OF AGRICULTURE. 

appearance appeared to be active, they were of smaller sizes, ranging 
from a pinhead to the size of a pea. Horses Nos. 110, 120, and 124 
were killed on the same day, although they had failed to show any 
indication of glanders by the eye test, which was also substantiated 
by the complement-fixation test with the blood of these animals. 
Post-mortem examination showed no signs of glanders in these animals. 

The final results are quite striking relative to the deficiency of 
immunization against glanders by killed bacteria. 

Of the remaining horses which were kept under observation, as 
indicated in Table 2, Nos. 117 and 119 died on October 17 and 25, 
respectively, of acute glanders after developing the cHnical form 
of the disease. No. 8G also showed indications of the disease in 
the early part of October. The final test on the remaining horses, 
namely, Nos. 86, 111, 121, and 123, was undertaken in the early 
part of January, 1914, when they were subjected to the ophthahnic 
and subcutaneous mallein tests and also to the complement-fixation 
and agglutination tests. All horses reacted to these different tests 
with the exception of No. 86, which reacted to the fixation, agglutina- 
tion, and ophthalmic tests but failed to react to the subcutaneous 
mallein test. Two days following the tests all the animals were 
destroyed and careful post-mortem examinations were made. The 
results showed glanders lesions in all animals, including No. 86, in 
varying degrees. The lungs in all cases contained numerous glanders 
nodules most of which were in an active stage, and in horse No, 86 
the apex of the left lung showed a typical glanderous pneumonia 
with the characteristic gelatinous infiltration and numerous nodules 
of various sizes throughout the remainder of the pulmonary tissue. 

It is interesting to note that all these vaccinated horses returned 
to the normal serum reaction of a negative case on or before the 
twelfth week after the vaccination, as may be seen from Table 4. 
The exposure in the corral was continued the same as during the 
entire course of the experiments and the weekly changes of stable 
exposure were also carried out. The appearance of the disease in 
these remaining animals seems to offer a more substantial basis for 
drawing conclusions as to the unsatisfactory results of these vaccina- 
tion tests. 

From our experience with outbreaks of glanders in stables, it 
appears that these experimental horses did not develop chnical 
manifestations of the disease in greater proportion than is the case 
with the average exposed horse. It is true that the exposure of the 
horses in the experiments was continuous although not unusually 
or unreasonably severe. Whether the horses which were included 
in this final test possessed a certain degree of immunity as a residt of 
the vaccination, during the period including the time between the first 
exposure and the last negative eye test, it is impossible to say; but 



IMMUNIZATION TESTS WITH OLANDERS VACCINE. 



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C-M 



IMMUNIZATION TESTS WITH GLANDERS VACCINE. 13 

from the sanitarian's standpoint this would be of theoretical impor- 
tance only, since even if such should be the case an immunit}^ of from 
two to four months could not be considered sufhcient for practical 
vaccination purposes. Furthermore, it should be remembered that 
some of these horses developed a latent form of the disease in 
less than three months from the last vaccination during the period in 
which the blood still contained the so-caUed immune bodies. 

CONCLUSIONS. 

The results obtained by these investigations appear to be sufficient 
to demonstrate the unsatisfactory results of this method of immuni- 
zation. Of the 13 immunized animals, 9 contracted the disease 
from natural exposure, which is a large proportion when it is consid- 
ered that all animals were aged and kept most of the time during 
the exposure out of doors. Of the 4 remaining immunized horses, 
1 died of impaction after the second vaccination, wliile the other 
3 animals were IdUed August 20, 1913, in order to ascertain by post 
mortem examination the possibiUty of glanders existing in these 
animals which had given positiA'C serum reaction, but which had 
returned to normal. In artificial infections of the vaccinated ani- 
mals they showed no resistance whatsoever, as both vaccinated 
horses promptly developed an acute form of the disease from touch- 
ing the Schneiderian membrane with a platinum loop v/hich had 
been touched to a growth of glanders bacilU. For the present, 
therefore, it seems ad^^sable to abstain from immunizing horses 
by this method, as a practice of this kind may do more harm than 
good. Owners ha^dng horses which are supposedly immunized 
would naturally become careless, thinking their animals were resistant 
to the disease, and thus even a better opportunity would be offered 
for the propagation of the disease than if the horses were not vac- 
cinated. Furthermore, the fact that the blood of vaccinated ani- 
mals can not be utihzed for serum tests for two or three months 
after the injections is also a great disadvantage in the eradication 
of the disease. 

As a result of this preUminary work it appears that the control 
and eradication of glanders must still be dependent upon the con- 
centration of our efforts in eliminating infected horses and the adop- 
tion of proper precautions against the introduction of infected ani- 
mals into stables free from the disease. The results achieved in 
Germany, Austria, and Canada by these methods have proved very 
encouraging, and no doubt if executed in the same spirit in this 
country a marked reduction in the cases of glanders would result. 



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